Final Exam Review - BCH IS 3201
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Questions and Answers

What is the primary use of Coomassie Brilliant Blue in protein analysis?

  • It specifically stains all proteins. (correct)
  • It increases the solubility of proteins.
  • It determines the size of protein fragments.
  • It measures the pH of the protein solution.

Which technique allows for the identification of a specific protein through antibody binding?

  • Polymerase Chain Reaction (PCR)
  • Western Blot (correct)
  • Protein Electrophoresis
  • Cell Culture Techniques

What does SDS-PAGE predominantly separate proteins based on?

  • Molecular weight alone (correct)
  • Acidity and alkalinity of proteins
  • Charge and shape of proteins
  • Native conformation and function

Why is a membrane blocked during a Western Blot procedure?

<p>To prevent unspecific binding of antibodies (C)</p> Signup and view all the answers

What is a key difference between native PAGE and SDS-PAGE?

<p>SDS-PAGE does not retain the protein's native size or shape. (C)</p> Signup and view all the answers

What role does EDTA play in the process described?

<p>It chelates divalent metal cations to protect DNA. (B)</p> Signup and view all the answers

What would likely happen if EDTA is omitted from the solution?

<p>The plasmid DNA would be destroyed by DNAses. (D)</p> Signup and view all the answers

What is the reason for using glucose in the lysis solution?

<p>To prevent premature lysis due to osmotic shock. (B)</p> Signup and view all the answers

Why is a buffer with alkaline pH (Tris pH=8) used in the process?

<p>To reduce electrostatic interactions between DNA and scaffolding proteins. (D)</p> Signup and view all the answers

What is the function of Sodium Acetate (pH=5.5) in the lysis solution?

<p>To neutralize the lysis buffer and aid in base pairing of DNA. (D)</p> Signup and view all the answers

What is the primary function of chlorophylls and carotenoids in the reaction center complex?

<p>To absorb light energy (B)</p> Signup and view all the answers

What process describes the formation of ATP in chloroplasts when light is present?

<p>Photo-phosphorylation (C)</p> Signup and view all the answers

Which molecule is reduced to NADPH during the process involving water and light energy?

<p>NADP (B)</p> Signup and view all the answers

Where in the chloroplasts are the photosystems primarily located?

<p>Thylakoid membranes (A)</p> Signup and view all the answers

What role do protons play in ATP generation within chloroplasts?

<p>They are pumped to create a proton gradient (C)</p> Signup and view all the answers

Which of the following is NOT a product of the light-dependent reactions of photosynthesis?

<p>Glucose (B)</p> Signup and view all the answers

What is the final electron acceptor in the photosynthetic electron transport chain?

<p>NADP (A)</p> Signup and view all the answers

Which component of the chloroplasts is responsible for proton pumping during ATP generation?

<p>Cytochrome b6f (D)</p> Signup and view all the answers

What is the formula used to make solutions from stock concentrations?

<p>c1V1 = c2V2 (D)</p> Signup and view all the answers

How is a 1% (weight/volume) solution generally defined?

<p>1 g of solute in 100 mL of solution (A)</p> Signup and view all the answers

What is the molarity of a solution that contains 0.5 moles of solute in 2 liters of solution?

<p>0.25 M (C)</p> Signup and view all the answers

If you need to prepare 240 mL of a 1X solution from a 15X stock solution, how much of the stock solution do you need to use?

<p>16 mL of 15X stock (B)</p> Signup and view all the answers

How many particles are there in 1 mole of a substance, according to Avogadro's number?

<p>6.02 x 10^23 (A)</p> Signup and view all the answers

What is the molecular weight (MW) of EDTA as given in the content?

<p>292 g/mol (D)</p> Signup and view all the answers

When creating a diluent for a concentration, what does a 2X solution mean?

<p>Double the concentration of the original solution (B)</p> Signup and view all the answers

If the formulation indicates to prepare 150 mL of a 25 mM solution of EDTA, how much EDTA is required?

<p>1.095 g (C)</p> Signup and view all the answers

Which type of DNA fragment runs faster during agarose gel electrophoresis?

<p>Supercoiled DNA (B), Smaller linear DNA fragments (D)</p> Signup and view all the answers

What is the role of Ethidium Bromide in gel electrophoresis?

<p>It intercalates between DNA base pairs and fluoresces under UV light (D)</p> Signup and view all the answers

Why is GelGreen considered safer than Ethidium Bromide?

<p>It cannot pass through cell membranes (D)</p> Signup and view all the answers

What type of enzyme is responsible for cleaving terminal residues from linear DNA?

<p>Exonuclease (C)</p> Signup and view all the answers

What is a characteristic of an isoschizomer?

<p>Recognizes the same sequence and cleaves at the same site (B)</p> Signup and view all the answers

What issue can result in off-site cleavage during a restriction digest?

<p>Star activity (C)</p> Signup and view all the answers

If plasmid X is 2000 bp and is digested with Enzyme 1 at the 500 bp site, what is the size of the resulting fragment?

<p>500 bp (B)</p> Signup and view all the answers

How many fragments would result when plasmid X is digested with both Enzyme 1 and Enzyme 2?

<p>Three (D)</p> Signup and view all the answers

What major difference exists between endonuclease and exonuclease enzymes?

<p>Endonuclease cleaves internal residues, while exonuclease cleaves terminal residues (D)</p> Signup and view all the answers

What does it indicate if an enzyme is described as a 'neoschizomer'?

<p>It recognizes the same sequence but cleaves at different sites (B)</p> Signup and view all the answers

What is the primary function of BamHI in the context provided?

<p>To cut DNA at specific sequences (C)</p> Signup and view all the answers

What does the notation '6538 bp' represent in the content?

<p>The total length of the plasmid pDP2 (D)</p> Signup and view all the answers

If BamHI cuts a fragment of 435 bp, what would be the approximate size of the remaining fragment after the cut?

<p>6103 bp (D)</p> Signup and view all the answers

What does the term 'star activity' refer to in the context given?

<p>Unexpected DNA fragment sizes (C)</p> Signup and view all the answers

What is the significance of the 'polyacrylamide gels' mentioned?

<p>They provide a medium for visualizing DNA fragments (C)</p> Signup and view all the answers

Which of the following is a potential issue when using restriction enzymes like BamHI?

<p>They may produce extra bands during electrophoresis (D)</p> Signup and view all the answers

What does 'linearizing the plasmid' accomplish in biotechnology?

<p>It allows for easy insertion of DNA fragments (D)</p> Signup and view all the answers

What can result from running an improper gel during DNA analysis?

<p>Decreased fragment visibility (C)</p> Signup and view all the answers

When three band fragments are formed during a restriction digest, what does this typically indicate?

<p>The presence of three different cut sites (C)</p> Signup and view all the answers

What role does the size marker play in gel electrophoresis?

<p>To determine the size of unknown DNA fragments (B)</p> Signup and view all the answers

Flashcards

EDTA's role in plasmid isolation

EDTA chelates divalent metal cations, preventing DNases from degrading plasmid DNA.

SDS in plasmid isolation

SDS disrupts cell membranes by interacting with their amphipathic phospholipid bilayers.

Effect of alkaline pH in plasmid isolation

Alkaline pH reduces electrostatic interactions between DNA and proteins, allowing DNA separation.

Impact of missing EDTA on plasmids

Without EDTA, DNases will degrade the plasmid DNA.

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Purpose of Sodium Acetate in plasmid isolation

Sodium Acetate neutralizes the alkaline lysis buffer and promotes DNA base pairing.

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Calculating solution concentration

Techniques to determine the relative amount of solute in a solution, using various expressions like weight/volume (w/v), volume/volume (v/v), and molarity.

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c1V1=c2V2 (Dilution Formula)

A formula used to determine the volume of a concentrated solution (c1, V1) needed to prepare a desired volume (V2) of a dilute solution (c2).

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Molarity (M)

A concentration unit that describes the number of moles of solute per liter of solution.

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Percent solutions (w/v, v/v)

Concentrations expressed as grams of solute per 100 milliliters (w/v) or volume of solute per 100 milliliters (v/v).

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X dilutions (1X, 2X etc)

Solutions with different concentrations, where the number 'X' represents the concentration- a 1X solution has a concentration that is one-time the 'standard' concentration.

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Stock solutions

Highly concentrated solutions of a substance used to prepare solutions of lower concentrations.

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EDTA

A chemical substance used in many solutions.

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Preparing solutions

Calculating amounts of solute and solvent to achieve a target concentration

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Linear DNA

A DNA molecule with two free ends, unlike circular DNA that forms a closed loop.

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Circular DNA

A DNA molecule that forms a closed loop, often found in plasmids and some viruses.

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Supercoiled DNA

A circular DNA molecule that is twisted upon itself, creating a compact structure.

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Agarose Gel Electrophoresis

A technique used to separate DNA fragments based on their size.

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Ethidium Bromide

A fluorescent dye that binds to DNA, making it visible under UV light.

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GelGreen

A safer alternative to Ethidium Bromide, also fluorescent and intercalating with DNA.

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Restriction Enzyme (Endonuclease)

An enzyme that cleaves DNA at specific sequences.

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Blunt Ends

Double-stranded DNA ends with flat ends, produced by some restriction enzymes.

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Sticky Ends

Double-stranded DNA ends with single-stranded overhangs, produced by some restriction enzymes.

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Isoschizomer

Restriction enzymes that recognize the same DNA sequence and cleave at the same site.

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What is the role of BamHI in the experiment?

BamHI is a restriction enzyme that cuts the plasmid DNA at a specific sequence, creating two fragments.

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What is the significance of the 435 bp fragment?

The 435 bp fragment is generated by the BamHI restriction enzyme and indicates successful digestion of the plasmid DNA.

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How does NDEL affect plasmid DNA?

NDEL is another restriction enzyme that cuts the plasmid DNA, creating a linear molecule. This linearization allows for the insertion of foreign DNA.

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What is the purpose of the 'stacked' gel in the experiment?

The stacked gel, containing both polyacrylamide and agarose, is used to separate DNA fragments by size. It allows visualization of different bands representing various fragments.

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Why are there extra bands in the linearization step?

The extra bands in the linearization step are likely due to 'star activity,' a phenomenon where restriction enzymes cut at sites outside their specific recognition sequence.

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How are fragments sized in the gel?

DNA fragments are separated based on their size and charge in the gel. Smaller fragments migrate further than larger fragments.

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What is the difference between polyacrylamide and agarose gels?

Polyacrylamide gels are used to separate smaller DNA fragments with higher resolution, while agarose gels are used for larger fragments.

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What are 'markers' in the gel?

Markers are DNA fragments of known sizes used as reference points to determine the sizes of unknown fragments.

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What are 'restriction enzymes' ?

Restriction enzymes act like molecular scissors to cut DNA at specific sequences, called recognition sites.

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SDS-PAGE

A technique used to separate proteins based on their molecular weight. Proteins are denatured with SDS and run through a gel under an electric field. Smaller proteins migrate faster.

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Native PAGE

A technique where proteins are separated based on their native (natural) size, shape, and charge. Proteins are not denatured, allowing for analysis of their native conformation.

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Western Blot

A technique to identify specific proteins in a sample. Separated proteins from SDS-PAGE are transferred to a membrane and probed with antibodies that bind to the target protein.

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Coomassie Brilliant Blue Staining

A dye that stains proteins, making them visible in a SDS-PAGE gel. It binds to proteins with a high affinity, allowing visualization.

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Why is protein size alone not enough to identify a protein?

Different proteins can have similar molecular weights, but they may have different amino acid sequences and biological functions. Antibody binding is needed to confirm the identity of a protein.

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Photosystems

Complexes within chloroplasts that capture light energy and convert it into chemical energy.

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Photophosphorylation

The process of generating ATP (energy currency) in chloroplasts using light energy.

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Where are Photosystems located?

Photosystems are located within the chloroplasts, specifically in the thylakoid membranes where light energy is captured.

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Role of Cytochrome b6f

Cytochrome b6f is a protein complex in chloroplasts responsible for transferring electrons and pumping protons across the thylakoid membrane.

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Generation of ATP

In chloroplasts, ATP is generated by the movement of protons across the thylakoid membrane, driven by the electron transport chain.

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Electron Acceptor

A molecule that accepts electrons during the electron transport chain in photosynthesis.

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Light Energy & Chlorophylls

Light energy is absorbed by chlorophyll molecules, which act as the primary light-absorbing pigments in photosynthesis.

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NADPH

A reduced electron carrier, produced in photosynthesis, that carries electrons and energy to fuel other reactions.

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Study Notes

Final Exam Review - BCH IS 3201

  • Topics needing review include lab math, plasmid digests, calculating fragment size, microarray slides, and using Bragg's law.

Lab Math

  • Making solutions from stock using c1V1=c2V2
  • % solutions (w/v and v/v)
  • Dilutions (1X, 2X, etc.)
  • Molarity calculations
  • Formula: (Formula weight) x (Volume desired) x (Molarity desired) = g needed
  • 1 mole = 6.02 x 10^23 particles

Practice Problems

  • Preparing 1L of 0.5M glucose solution (MW 180.2 g/mol)
  • Preparing 1L of 100 mM glucose solution (MW 180.2 g/mol)
  • Preparing 10mL of 1M glycine solution (MW 75.07 g/mol)
  • Preparing 10mL of 0.5M glucose solution (MW 180.2 g/mol)
  • Calculating final concentration of 1M glycine in 10mL with 9mL water
  • Calculating concentrations of 10mg glucose / 100mL water and 100mL of 2% alanine (89.09 g/mol)
  • Making 200mL of 20mM phosphate buffer from 0.5M stock
  • Making 1L of 50% (w/v) glucose solution (MW 180.2 g/mol)

Pipetting

  • Minimum range on a pipettor is determined as 10% of the maximum range
  • Pipettor ranges (ul)
    • P10: 0-10 (1-10)
    • P20: 0-20 (2-20)
    • P200: 0-200 (20-200)
    • P1000: 0-1000 (100-1000)

Plasmid Purification - Reaction Components

  • EDTA chelates divalent metal cations—preventing DNAse degradation of plasmid DNA
  • Glucose prevents instant cell lysis by osmotic shock
  • Alkaline pH (Tris pH 8) reduces electrostatic interactions between DNA and scaffolding proteins

Plasmid Purification

  • Examining why plasmid DNA renatures and chromosomal DNA does not: plasmid DNA is usually smaller/topologically intertwined than chromosomal DNA
  • Examining the use of 90% ethanol as a DNA precipitant
  • Discussing the purpose of 70% ethanol.

Agarose Gel Electrophoresis

  • Smaller DNA fragments move faster in agarose gel electrophoresis
  • Ethidium Bromide and GelGreen: fluorescent dyes intercalating between base pairs for visualization under UV light
  • GelGreen is safer than EtBr

Restriction Enzymes

  • Restriction enzymes cleave terminal or internal residues from linear DNA, recognizing specific DNA sequences and cleaving at the same or different sites
  • Different conditions can cause off-site cleavage (star activity).

Restriction Digest

  • Enzyme 1 cleaves at 500 bp
  • Enzyme 2 cleaves at 1000bp site

Question - Digesting plasmid Q with Baml and Hindl

  • Lane 3: Incomplete digest bands
  • Lane 4: Incomplete digest + star activity

Plasmid Digest - Practice

  • pBLU plasmid with BamHI - 5437bp fragment
  • pBLU plasmid with XmnI and NdeI - different sizes of fragments may be possible.

Question - Digesting Lambda Phage DNA

  • Digest with BamHl: various fragment sizes will be created, totalizing 48,502 base pairs
  • Digest without completing: various additional fragments, some with variable, incomplete digest bands

Question - Cutting pUC19 with Restriction Enzymes

  • How many bands will result from digestion with specific restriction enzymes on a gel

Polyacrylamide Gels

  • Resolve proteins or DNA fragments with similar sizes
  • Polyacrylamide gels are created with crosslinked acrylamide and bisacryamide
  • Native gels separate proteins by size, shape, and charge
  • SDS-PAGE separates proteins based on molecular weight.

Native Gel System

  • Non-denaturing - protein maintains its natural structure
  • Migration depends on charge and size
  • Proteins stable post-extraction

SDS Gel System

  • Denaturing and reducing - protein unfolds and loses its original form
  • Migration only depends on mass (molecular weight)

Western Blot

  • Detecting the protein of interest using BLOTTO (milk protein) to block the membrane and antibodies to bind to the target protein
  • Identifying specific targets

Polyacrylamide Gel Electrophoresis (PAGE)

  • SDS (detergent) coats proteins with uniform negative charge; disrupts protein native structure, and allows migration based on size
  • B-mercaptoethanol helps reduce disulfide bridges

Kinetics

  • enzyme speed-up rates
  • reaction rate (v0)
  • substrate concentration ([S])
  • Michaelis-Menten equation: V0 = (Vmax * [S])/(Km + [S])
  • Vmax: maximum initial reaction rate
  • Km: Michaelis constant, substrate concentration at which v = Vmax/2.

Double Reciprocal Plot (Lineweaver-Burk Plot)

  • To determine Vmax and Km more precisely.
  • The slope = Km/Vmax
  • Y-intercept = 1/Vmax
  • X-intercept = -1/Km

Types of Inhibition Kinetics

  • Competitive, mixed, and uncompetitive inhibition affects both Km and Vmax in different ways.

Additional Questions

  • Determining values and interpretations of data from the given experimental conditions

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BCHS 3201 Final Exam Review Fall 2022 PDF

Description

Prepare for your BCH IS 3201 final exam with this comprehensive review covering essential lab math topics such as solution preparation, dilutions, and molarity calculations. You will also encounter practice problems related to making various concentrations and solutions. Test your understanding and ensure you're ready for the exam!

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